Leveraging the Power of In Vitro : An Exploration of Applications, Challenges and Solutions

Rob Brazas, Ph.D. Senior Product Manager, Lucigen September, 2017

www.lucigen.com Agenda Leveraging In Vitro Transcription for Your Success

• Overview of in vitro transcription (IVT) technology • Applications of IVT-produced RNA – Overview of each type of application – Examples of IVT use for each application with data from the literature • Key challenges of IVT and working with RNA • Solutions to those challenges – AmpliScribe™ T7-Flash Transcription Kit – DuraScribe® T7 Transcription Kit In Vitro Transcription (IVT) Transcription Generally Starts with a dsDNA Template

In Vitro Transcription: Production of RNA in a tube (e.g. microfuge tube) using an RNA polymerase (usually phage derived), ribonucleotides and appropriate buffer conditions.

DNA of interest (e.g. ORF, shRNA, aptamer) dsDNA 5’ 3’ Template 3’ T7 Promoter 5’ SP6 Promoter T3 Promoter IVT Template Options Multiple Forms of DNA Make Acceptable IVT Templates

Blunt End 5’ 3’ Linearized 3’ T7p 5’

5’ 3’ Linearized Plasmid 3’ T7p 5’ 5’ Overhang

5’ 3’ PCR Product 3’ T7p 5’

+1 5’ 3’ 5’ TAATACGACTCACTATAG Synthetic Construct 3’ T7p 5’ 3’ ATTATGCTGAGTGATATCCCNNNNNNNNNNNNN 5’ T7 Promoter

2nd Strand cDNA 5’ AAAA 3’ cDNA 3’ TTTT T7p 5’ 1st Strand cDNA

Caution: 3’ overhangs may lead to the production of spurious RNA transcripts IVT Reactions Production of Large Amounts of RNA from ~1 µg of DNA

T7 RNA Polymerase + ATP, UTP, GTP & CTP, reaction buffer, +/- RNase Inhibitor

DNA of interest (e.g. ORF, shRNA, aptamer) dsDNA 5’ 3’ Template 3’ T7 Promoter 5’

T7 RNA Polymerase 5’ 3’ 3’ T7 Promoter 5’

T7 RNA Polymerase 5’ T7 RNA 5’ Polymerase 3’ 3’ T7 Promoter 5’

IVT RNA transcripts 5’ 3’ Production of Modified or Labeled IVT RNA Modified or Labeled NTPs are Effectively Incorporated

+ Reaction buffer T7 RNA +/- RNase Inhibitor Polymerase + ATP, UTP, GTP & CTP + Labeled or modified ribonucleotides (e.g. Biotin-UTP, Digoxigenin-UTP, 2’-F-UTP*)

5’ 3’ 3’ T7 Promoter 5’

Biotin Biotin Biotin Biotin-RNA U U U U U

Dig Dig Dig

Digoxigenin-RNA U U U U U

2’-F-RNA* U UF U UF UF

* Incorporation of 2’-F-UTP and 2’-F-CTP requires a mutated T7 RNA Polymerase Note: It is also possible to incorporate radiolabeled ribonucleotides Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible

 Viral RNA synthesis and to start replication  mRNA synthesis for in vitro and in vivo applications  RNA structure studies  RNA aptamer synthesis for SELEX  dsRNA or shRNA synthesis for RNAi  CRISPR gRNA synthesis for RNP-mediated gene editing  Riboprobe synthesis for Northern blotting, in situ hybridization  RNA amplification → amplified cDNA (MessageBOOSTER™ Kits)  miRNA synthesis  Anti-sense RNA synthesis  More… you’re only limited by your imagination! Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible

 Viral RNA synthesis and transfection to start replication  mRNA synthesis for in vitro and in vivo applications  RNA structure studies  RNA aptamer synthesis for SELEX  dsRNA or shRNA synthesis for RNAi  CRISPR gRNA synthesis for RNP-mediated gene editing  Riboprobe synthesis for Northern blotting, in situ hybridization  RNA amplification → amplified cDNA (MessageBOOSTER™ Kits)  miRNA synthesis  Anti-sense RNA synthesis  More… you’re only limited by your imagination! Flaviviradae Family Great Models for All Things Related to IVT RNA

Flaviviridae Example: Hepatitis C

IRES

From: Moradpour, Penin and Rice (2007) Replication of Hepatitis C virus. Reviews | Microbiology 5:453-463

Hepatitis C Virus • 9.6 kb +strand RNA genome – Flaviviridae family (e.g. Yellow fever, Dengue) • After cellular entry, translates genomic RNA into a polyprotein using IRES to initiate • Polyprotein is cleaved by cellular protease (diamonds) and viral proteases (arrows) o Proteolysis occurs co- and post-translationally Hepatitis C Virus Replication Cycle Replication Starts with of the Incoming Genome

From: Moradpour, Penin and Rice (2007) Replication of Hepatitis C virus. Nature Reviews | Microbiology 5:453-463

a) Cell binding and entry d) RNA genome replication (+RNA) → (-) RNA → (+) RNA b) Uncoating and genome release e) Packaging and assembly c) IRES-mediated translation and polyprotein processing f) Maturation and release Translation of IVT RNA Uncapped IVT are Poorly Translated

T7 RNA Polymerase 5’ 3’ T7 Promoter ORF 5’

Poor translation, Uncapped mRNA unstable + Post-transcriptional PolyA Tailing Poor translation, Uncapped/PolyA+ mRNA A n improved stability Translation of IVT RNA Addition of a m7-G Cap Analog Improves Translation

T7 RNA 7 7 Polymerase 5’-m G Cap Analog (m G(5')ppp(5')G) added to IVT reaction 5’ 3’ T7 Promoter ORF 5’

Improved translation, Capped mRNA m7-G unstable + Post-transcriptional PolyA Tailing Improved translation & Cap/Poly(A) mRNA m7-G A n stability

Cap Analogs (ARCA, Normal) Note: 5’ Caps can also be added 3’ post-transcriptionally

(OH) Translation of IVT RNA Viral IRES Enhance Translation in the Absence of a Cap

IRES = Internal Ribosome Entry Site

T7 RNA Polymerase 5’ 3’ 3’ T7 Promoter IRES ORF 5’

Improved IRES - mRNA 5’ 3’ translation Translation of IVT RNA IVT mRNA can be Translated In Vivo and In Vitro

me-G An  Cell-free expression  Transfection into cultured cells • Rabbit reticulocyte extracts  Microinjection (e.g. oocytes) • Wheat germ extracts • HeLa extracts

Benefits Benefits • Speed • Transient/short term expression • No cellular toxicity • Pulse of expression • Better solubility • No potential DNA effects Applications Applications • Protein:protein interactions • RNA processing, stability studies • Structure studies • Protein expression • Enzyme/functional assays o e.g. CRISPR nucleases Transfection of YFV, HCV Viral RNAs Made In Vitro Transfected RNAs are Translated and Initiate Replication

T7p YFV Replicon with Luciferase ORF T7p HCV Replicon with NeoR ORF In vitro transcription In vitro transcription

5’ 3’ 5’ 3’ Transfection, growth, Transfection, growth, dilution & luciferase assay replating, colony staining (-) control (+) control

From: Gonzalez et. al. (2007) Selection of an optimal RNA transfection reagent and comparison to for the delivery of viral RNA. Journal of Virological Methods. 145:14-21 Using In Vitro Transcription and Translation to Identify HCV Protein: Host Protein Interactions by GST Pull-downs

T7p HCV NS5B ORF In vitro transcription 5’ 3’ From: Domitrovic et. al. (2005) Role of La autoantigen and polypyrimidine tract-binding In vitro translation – RRL protein in HCV replication. Virology. 335:72-86 +35S-methionine

B GST + NS5B Mix, bind, B GST La NS5B then wash + B GST PTB + NS5B

B GST B GST La Electrophorese on SDS-PAGE 35 B GST PTB NS5B and image S-signals Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible

 Viral RNA synthesis and transfection to start replication  mRNA synthesis for in vitro and in vivo applications  RNA structure studies  RNA aptamer synthesis for SELEX  dsRNA or shRNA synthesis for RNAi  CRISPR gRNA synthesis for RNP-mediated gene editing  Riboprobe synthesis for Northern blotting, in situ hybridization  RNA amplification → amplified cDNA (MessageBOOSTER™ Kits)  miRNA synthesis  Anti-sense RNA synthesis  More… you’re only limited by your imagination! Using In Vitro Transcribed HCV IRES RNA to Determine Its Crystal Structure

T7p HCV IRES Central Domain HDV Ribozyme In vitro transcription 5’ 3’ From: Berry et. al. (2011) Crystal Structure of the HDV Ribozyme cleavage HCV IRES Central Domain Reveals Strategy for Start- Codon Positioning. Cell. 19:1456-1466 5’ 3’

5’ 3’ HCV IRES Central Core Cleanup, crystallization, crystallography Central Domain Structure Model of IRES on 40S Ribosomal Subunit Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible

 Viral RNA synthesis and transfection to start replication  mRNA synthesis for in vitro and in vivo applications  RNA structure studies  RNA aptamer synthesis for SELEX  dsRNA or shRNA synthesis for RNAi  CRISPR gRNA synthesis for RNP-mediated gene editing  Riboprobe synthesis for Northern blotting, in situ hybridization  RNA amplification → amplified cDNA (MessageBOOSTER™ Kits)  miRNA synthesis  Anti-sense RNA synthesis  More… you’re only limited by your imagination! What Are RNA Aptamers? Think of Them as RNA-based Antibodies

ssRNA Aptamer with Predicted Single-stranded RNA molecules Secondary Structure that: • Fold into RNA structure with secondary and tertiary structure • Generally 40 to 100 long

• Correct sequences/structures Folds into 3D structure that tightly and bind tightly and specifically to a specifically binds its target target molecule (e.g., protein, toxin, cell receptor, other RNAs…)

Think “Antibody made of RNA”… How Are Good RNA Aptamers Made and Identified? SELEX Systematic Evolution of Ligands by EXponential Enrichment

T7p A Random B Chemical synthesis of selected aptamer dsDNA Library Sequence library In vitro transcription Target

Binding

dsDNA

PCR 8 – 15X

3’ 5’ Washing 3’ 5’ cDNA

Reverse transcription

Elution Identification of Aptamers that Bind HCV NS5B Replicase Used Both Standard RNA and 2’-F-CTP, -UTP Substituted RNA

Standard RNA Aptamers Identified

R-OH

Aptamer:NS5B Gel Shift Binding Assay Experimental Design • Used synthetic DNA library with T7 promoter to synthesize RNA aptamer library • Used immobilized NS5B replicase as target • Identified (4) standard RNA aptamers • Demonstrated inhibition of HCV replication

From: Lee et. al. (2013) Inhibition of Hepatitis C Virus (HCV) Replication by Specific RNA Aptamers against HCV NS5B RNA Replicase. J. Virol. 87:7064–7074 Identification of Aptamers that Bind HCV NS5B Replicase Used Both Standard RNA and 2’-F-CTP, -UTP Substituted RNA

Experimental Design • Same experimental design but used DuraScribe® T7 Transcription Kit to make RNase A- resistant aptamers • Durascribe® Kits generate 2’-F-UTP, -CTP substituted RNAs (aptamers here) • Identified one high affinity 2’-F-Aptamer, R-F Full-Length & Truncated R-F Aptamers • Also inhibited HCV replication

Inhibition of an HCV Replicon

From: Lee et. al. (2013) Inhibition of Hepatitis C Virus Original (HCV) Replication by Specific RNA Aptamers against HCV NS5B RNA Replicase. J. Virol. 87:7064–7074 Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible

 Viral RNA synthesis and transfection to start replication  mRNA synthesis for in vitro and in vivo applications  RNA structure studies  RNA aptamer synthesis for SELEX  dsRNA or shRNA synthesis for RNAi  CRISPR gRNA synthesis for RNP-mediated gene editing  Riboprobe synthesis for Northern blotting, in situ hybridization  RNA amplification → amplified cDNA (MessageBOOSTER™ Kits)  miRNA synthesis  Anti-sense RNA synthesis  More… you’re only limited by your imagination! RNA Interference using shRNAs Targeting HCV IVT shRNAs Induced RNAi and mRNA Cleavage

Experimental Design shRNA Template(s) • Designed shRNAs targeting the HCV IRES In vitro transcription • Transcribed those shRNAs using the AmpliScribe™ T7-FLASH Transcription Kit IRES • Cotransfected shRNA into cells transfected pHCV Firefly with HCV IRES-driven luciferase reporter Luciferase plasmid (+control)

Co-transfection Inhibition of a pHCV Luciferase Expression

Determine luciferase activity

From: Vlassov et. al. (2007) shRNAs Targeting Hepatitis C: Effects of Sequence and Structural Features, and Comparison with siRNA. Oligonucleotides 17:223–236 Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible

 Viral RNA synthesis and transfection to start replication  mRNA synthesis for in vitro and in vivo applications  RNA structure studies  RNA aptamer synthesis for SELEX  dsRNA or shRNA synthesis for RNAi  CRISPR gRNA synthesis for RNP-mediated gene editing  Riboprobe synthesis for Northern blotting, in situ hybridization  RNA amplification → amplified cDNA (MessageBOOSTER™ Kits)  miRNA synthesis  Anti-sense RNA synthesis  More… you’re only limited by your imagination! CRISPR Gene Editing by RNP Delivery Synthesis of Guide RNAs by IVT

Clone, synthesize gRNA Expression Construct

Deliver to Cells 5’ T7p gRNA 3’ 3’ 5’ Transfection In vitro transcription Electroporation gRNA Biolistic Gun Cas9 WT, Detect Form RNP Cas9 D10A or Edited Cells Complex Cpf1 Cas9 WT, Co-deliver ssDNAs and Cas9 D10A or Cpf1 dsDNAs to drive specific mutations CRISPR Gene Editing by RNP Delivery Synthesis of Guide RNAs by IVT Common Mutations

Experimental Design • Designed gRNAs targeting 4 different genes in maize • Transcribed those gRNAs using the AmpliScribe™ T7- FLASH Transcription Kit • Complexed with Cas9 protein and delivered to maize embyros by biolistic particle transformation • Amplified target regions and sequenced amplicons to identify mutation frequencies & changes • Also, co-delivered ssDNA “repair oligo” to change ALS2 proline 165 to a serine and grew up edited plants

From: Svitashev et. al. (2016) Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat. Comm. 7:13274 CRISPR Gene Editing by RNP Delivery Generating Herbicide Resistant Plants

Guide RNA to ALS Gene and “Helper Repair Oligo” Test Plants Treated with Herbicide

WT eP165S

• Proline to serine mutation confers herbicide resistance • Right plant: Edited plant, herbicide resistant • Left plant: Wild type, herbicide sensitive

From: Svitashev et. al. (2016) Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat. Comm. 7:13274 Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible

 Viral RNA synthesis and transfection to start replication  mRNA synthesis for in vitro and in vivo applications  RNA structure studies  RNA aptamer synthesis for SELEX  dsRNA or shRNA synthesis for RNAi  CRISPR gRNA synthesis for RNP-mediated gene editing  Riboprobe synthesis for Northern blotting, in situ hybridization  RNA amplification → amplified cDNA (MessageBOOSTER™ Kits)  miRNA synthesis  Anti-sense RNA synthesis  More… you’re only limited by your imagination! Northern Blotting with a Dig-labeled Riboprobe Determining Expression Levels of a New Cellular Transcript

Experimental Design • Ran total RNA on formaldehyde gel DipA mRNA • Transferred RNA to a membrane • Hybridized antisense Dig-DIPA riboprobe 5’ 3’ 5’ An • Washed and probed with alkaline Antisense DIPA Dig Dig Dig Dig-riboprobe phosphatase conjugated anti-dig antibody • Washed and detected bound antibody (DIPA + chemiluminescent mRNA) with chemiluminescent substrate and AP substrate film!

Northern Blot Results

Brazas and Ganem (1996) Science 274:90-94 Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible

 Viral RNA synthesis and transfection to start replication  mRNA synthesis for in vitro and in vivo applications  RNA structure studies  RNA aptamer synthesis for SELEX  dsRNA or shRNA synthesis for RNAi  CRISPR gRNA synthesis for RNP-mediated gene editing  Riboprobe synthesis for Northern blotting, ISH  RNA amplification → amplified cDNA (MessageBOOSTER™ Kits)  miRNA synthesis  Anti-sense RNA synthesis  More… you’re only limited by your imagination! Using IVT to Amplify RNA in Limiting Samples Produce Amplified cDNA Without Altering mRNA Profiles

MessageBOOSTER™ qPCR Kit Protocol

Poly(A) mRNA 5’ AAAA3’ • Linear RNA amplification st process preserves Round 1 1 -strand TTTT T7p-Oligo(dT) Primer + RT cDNA Synthesis transcripts relative 5’ AAAA 3’ abundance 3’ TTTT Round 1 2nd-strand RNase H, Random • Perform more RT-qPCR cDNA Synthesis Hexamers + RT reactions from precious 5’ AAAA samples 3’ TTTT T7p Amplification - In T7 RNA Polymerase + NTPs • Readily and reproducibly Vitro Transcription detect even low- 3’ UUUU 5’ abundance transcripts in Round 2 1st-strand Random Hexamers +RT RNA from a single cell cDNA Synthesis 5’ AAAA 3’ • Produce enough cDNA to 3’ UUUU 5’ archive for later use RNase H Treatment

5’ AAAA 3’ RNA = RED cDNA (amplified) DNA = GREEN Key Challenges Associated with In Vitro Transcription Time and Yield Are the Main Challenges

• Time o Many IVT reactions take 2-3 hours to produce maximal RNA yields • Yield o Low yield kits/protocols mean more repeat IVT reactions o High yield kits decrease the number of reactions needed to produce enough RNA – decreasing costs and time spent • RNA degradation o RNA is very sensitive to degradation by RNases! o Some of those applications require multiple steps and present many opportunities for RNases to degrade your precious RNA (e.g SELEX) • Synthesizing both long and short RNAs o Transcribing full-length long RNAs can be difficult due to secondary structure, GC content… (e.g. critical for RNA virus genome studies) o Short RNAs can also be difficult to produce due to minimal length of templates AmpliScribe™ T7-FLASH Transcription Kit Solves the Time and Yield Challenges of IVT

• Fast o Produce maximal RNA yields in only 30 minutes. • High Yields o Produce 180 μg of RNA from only 1 μg of template DNA. • Flexible o Accepts a variety of template with T7 promoters including linearized vectors, PCR products, cDNA, and dsDNA oligos. • Scalable o Reactions can be scaled up to quickly produce milligram - gram amounts of RNA. • Multi-application compatible o Make long (9 kb in house, >11 kb by researchers) o Make short (>25 bases) RNA transcripts o Suitable for all the applications discussed today and more AmpliScribe™ T7-FLASH Transcription Kit Higher Yield than Competitor Kits in Only 30 Minutes

Higher Yields in 30 Minutes vs 60 High Yields from Both Small and Minutes with Competitor Kits Large Templates

• Various template sizes • 1.4 kb template • 1 µg of template DNA/rxn • 1 µg of template DNA/rxn • 30 minute reactions AmpliScribe™ T7-FLASH Transcription Kit High Yields of Small RNA or from Limiting Template Input

High Yields of Very Short Yields Remain High With Transcripts (>26 bases) Decreasing Template Amounts

RNA RNA Yield RNA Yield Transcript Size (µg) (pmoles) 26 bases 12 1319

47 bases 24 1459

96 bases 36 1071

335 bases 76 648

1.4 kb 176 359

6 kb 187 89

• Various template sizes • 1 µg of template DNA/rxn • 30 minute reactions • Various template amounts Note: Increasing rxn temp, time, & template • 1.4 kb transcript amounts increases short transcript yields • 30 minute reactions DuraScribe® T7 Transcription Kit RNase A-resistant Transcripts for Demanding Applications

• Stable o DuraScript® RNA transcripts are fully resistant to RNase A o Protects RNA during complicated experiments like SELEX or in harsh environments like tissue culture media • High Yields o Produce ~40 - 60 μg of DuraScript RNA from only 1 μg of a 1.4 kb template in 4 – 6 hours o 110 – 307 pmol of transcripts depending on the length • Flexible o Uses the same T7 promoters and templates as standard T7 RNA polymerase (e.g. AmpliScribe™ Kits) • Multi-application compatible o Make long or short transcripts o Creates suitable substrates for RNAi, (dsRNA for dicing), antisense RNA, miRNA and aptamer identification o Not suitable for experiments requiring translation How Does the DuraScribe® T7 Transcription Kit Work Incorporates 2’-F-CTP & 2’-F-UTP with a Modified T7 RNAP

DuraScribe® T7 RNA Polymerase: Modified T7 RNA polymerase that readily incorporates modified nucleotides

RNase A-resistant RNA Transcripts

Transcription DuraScribe T7 RNAP DuraScribe T7 Promoter T7 Promoter T7 RNAP DuraScribe® T7 Transcription Kit Produce Stable, RNase A-resistant Transcripts

Resistant to RNase A DuraScript RNA is Stable in Tissue DuraScript Culture Media for ≥2 Hours RNA RNA RNase A: - + - + RNA DuraScript RNA Tissue Culture Media (min.): - 15 - 15 30 60 120

• 1.4 kb transcripts (standard, DuraScript®) • 1.4 kb transcripts (standard, DuraScript®) • +/- 100 µL D-MEM + 10% FCS @37°C • +/- 1 unit high purity RNase A • Incubated for indicated times and then • 30 minute incubation, then gel analysis analyzed by gel electrophoresis DuraScribe® T7 Transcription Kit DuraScript® dsRNA are Effective RNAi Substrates

DuraScript dsRNA is Digested by Recombinant Human Dicer Enzyme as Efficiently as Canonical dsRNA 800 bp 800 bp Canonical DuraScript Actin Actin dsRNA dsRNA Dicer: - + - +

• 800 bp dsRNA transcripts (standard, DuraScript®) • 30 µg each RNA digested overnight at 37°C with 30 U of recombinant human dicer enzyme • Diced RNAs were purified by and analyzed by non-denaturing PAGE DuraScribe® T7 Transcription Kit DuraScript® dsRNA are Effective RNAi Substrates

High Yields of RNase A-resistant DuraScript RNA

• Each reaction used 1 µg of template (templates = same construct digested at 4 locations to produce different length transcripts) • Reactions were incubated at 37°C for 4 hr and quantified Summary IVT Offers a Rapid, Cost-Effective Method of Making RNA for a Wide Variety of Applications

• IVT RNA can be used for multiple applications from in vitro or in vivo protein expression through RNAi knockdowns and CRISPR gene editing. • Key challenges include long IVT reaction times, low yields and RNA degradation • The AmpliScribe™ Kit overcomes the first two challenges with the fastest reactions (30 min) and the highest yields • The DuraScribe® Kit offers the unique ability to produce RNase-A resistant RNA – Optimal for: o Complicated experiments such as SELEX-based aptamer identification o Use in harsh environment where degradation may affect your results (e.g. tissue culture) Questions? www.lucigen.com

Lucigen Tech Support Contact me. [email protected] Rob Brazas, Ph.D. (608) 831-9011 Sr. Product Manager 8 am – 5 pm central time [email protected]

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